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United States Patent |
5,789,183
|
Lee
,   et al.
|
August 4, 1998
|
Serological detection and identification of rice blast
Abstract
Monoclonal antibodies (MAbs) useful in the serological detection and
identification of rice blast were produced by hybridoma cells formed from
fusions of myeloma cells with splenocytes from BALB/c mice immunized with
an extract of a liquid culture fluid of an isolate of Pyricularia grisea
race IB-49. These MAbs reacted similarly with the antigens in various
serological techniques used, and did not cross react with any unrelated
fungal isolates representing 11 genera, but reacted positively with all 20
races or isolates of P. grisea. The MAbs could detect homologous antigen
at about 60 ng fungal protein/ml and a 5-fold dilution of the extracts of
infected rice tissue by ELISA. In accordance with another embodiment of
the present invention, hybridoma lines secreting antibodies positive for
the immunogen and negative for healthy rice tissue were selected from
three independent fusions of NS-1 myeloma cells with splenocytes from mice
immunized with crushed conidial suspensions of P. grisea race IB-49. MAbs
secreted from cell line 4G11, deposited with ATCC as HB11178, reacted
strongly with conidial antigen. In cross-reaction tests with ELISA, MAb
4G11 reacted negatively with isolates representing 11 fungal genera and
reacted positively with 11 and 12 isolates of P. grisea in ELISA and IFA,
respectively. MAb 4G11 could detect homologous conidial antigen at 14-70
ng/ml, 10-20 conidia/well, and the fungal antigen in infected rice tissue
in ELISA.
Inventors:
|
Lee; Fleet N. (Stuttgart, AR);
Scott; Howard A. (Fayetteville, AR);
Xia; Jun Q. (Fayetteville, AR)
|
Assignee:
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University of Arkansas (Little Rock, AK)
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Appl. No.:
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071573 |
Filed:
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June 1, 1993 |
Current U.S. Class: |
435/7.31; 435/7.92; 435/70.21; 435/287.2; 435/288.4; 435/341; 435/911; 435/975; 436/518; 436/548; 530/388.5 |
Intern'l Class: |
G01N 033/569; G01N 033/543 |
Field of Search: |
435/7.31,7.92,911,975,242,291,70.21,172.2,240.27,288.4,287.2
530/388.5
436/548,518
935/104,106
|
References Cited
U.S. Patent Documents
4376110 | Mar., 1983 | David et al. | 435/7.
|
4693968 | Sep., 1987 | Kitagawa | 435/975.
|
4803155 | Feb., 1989 | Petersen et al. | 435/7.
|
4845197 | Jul., 1989 | Petersen et al. | 435/7.
|
4879217 | Nov., 1989 | Petersen et al. | 435/7.
|
5047207 | Sep., 1991 | Lankow et al. | 422/58.
|
5118610 | Jun., 1992 | Kitto et al. | 435/7.
|
5120834 | Jun., 1992 | Gargan et al. | 530/388.
|
5124264 | Jun., 1992 | Imura et al. | 530/388.
|
Foreign Patent Documents |
0135378 | Mar., 1985 | EP.
| |
137403 | May., 1979 | DE.
| |
Other References
Xia, 1991, Development and Characterization of Monoclonal Antibodies
Specific for Antigens of the Rice Blast Fungus, Pyriclaria grisea, Ph.D.
Thesis, University of Arkansas Dissertation Abstracts International, Order
#AAD92-37414, Abstract.
Xia et al, 1990, Monoclonal Antibodies to a Saline Extractable Antigen
Associated with Conidia of Pyricularior oxyzae, Race IB-49, Phytopathol
80:1048, Abstract A725.
Xia et al, 1991, Monoclonal Antibodies Specific for Conidia of the Rice
Blast Fungus, Phytopathol 81:1136, Abstract 10.
Kitagawa et al, 1987, A Novel Enzyme Immunoassay Commonly Applied for Ten
Strains of Pyricularia oxyzae, Microbiol. Immunol. 31:1197-1207.
Dewey et al, 1991 (Mar.), Antibodies in Plant Science, Acta Bot. Neerl.
40(1):1-27.
Burge et al, 1990, Bioaerosola: Prevaline and Health Effects in the Indoor
Environment, J. Allergy Clin. Immunol. 86:687-701.
D'Amato et al, 1989, Allergenic Pollens in the Southern Mediterranean Area,
J. Allergy Clin Immunol 83:116-22.
Hellstrom et al, 1985, In "Monoclonal Antibodies for Cancer Detection and
Therapy", (R.W. Baldwin et al, eds). Academic Press, London, p. 20.
Goding, 1983, Monoclonal Antibodies: Principles and Practice Academic
Press, London, pp. 56-97.
Abstract 591, Phytopathology, vol. 81, Oct., 1991, p. 1212,
Characterization of Monoclonal Antibodies For Conidial Antigens of
Pyricularia grisea Sacc.
Development of Monoclonal Antibodies to a Liquid Culture Extract of
Pyricularia grisea, J.Q. Xia, F.N. Lee, and K.S. Kim, Dept. of Plant
Pathology, University of Arkansas, AR 72701.
DNA Fingerprint (MGR) Analysis of Two Local Rice Blast Populations in
Arkansas, J.Q. Xia, J.C. Correll, F.N. Lee and D.D. Rhoads. Dept. of Plant
Pathology and Dept. of Biological Sciences, Univ. of Arkansas,
Fayetteville, Arkansas AR 72701.
|
Primary Examiner: Chan; Christina Y.
Assistant Examiner: Grun; James L.
Attorney, Agent or Firm: Alexander; Daniel R.
Head, Johnson & Kachigian
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application is a continuation of application Ser. No. 07/930,239,
filed Aug. 14, 1992, now abandoned.
Claims
What is claimed as invention is:
1. A method of serological detection and identification of Pyricularia
grisea comprising the steps of:
producing monoclonal antibodies specific for and highly reactive with
antigens of Pyricularia grisea race IB-49 which infect rice plants, highly
reactive for Pyricularia grisea race IB-49 infected rice tissue, and
substantially unreactive with healthy rice plant tissue from fusions of
myeloma cells with splenocytes from mice immunized with an antigen
comprising an extract of a liquid culture of Pyricularia grisea race
IB-49;
reacting the monoclonal antibodies with a sample of rice tissue, spores or
extract thereof; and
measuring the specific binding reaction between the monoclonal antibodies
and Pyricularia grisea antigen in said sample to determine the presence of
Pyricularia grisea.
2. In a method of detecting Pyricularia grisea in rice plants including the
steps of producing a sample to be tested from the rice plants, reacting
the sample with monoclonal antibodies specific for Pyricularia grisea, and
measuring the specific binding reaction therebetween to determine the
presence or quantity of Pyricularia grisea present in the sample, the
improvement comprising reacting with the sample antibodies which are
highly reactive with Pyricularia grisea race IB-49 which infect rice
plants, highly reactive with Pyricularia grisea race IB-49 infected rice
plant tissue, and substantially unreactive with healthy rice plant tissue
or other fungal genera and produced from hybridoma cells having the
identifying characteristics of hybridoma cell line 4G11 having ATCC
deposit HB11178 and antibody-producing reclones thereof.
3. A method of serological detection and identification of Pyricularia
grisea comprising the steps of:
producing monoclonal antibodies specific for and highly reactive with
antigens of rice-pathogenic Pyricularia grisea race IB-49, highly reactive
with Pyricularia grisea race IB-49 infected rice tissue, substantially
unreactive with healthy rice plant tissue, and weakly reactive or
unreactive with rice- or grass-pathogenic fungal genera other than
Pyricularia from fusions of myeloma cells with splenocytes from mice
immunized with an antigen from an isolate of Pyricularia grisea race
IB-49, wherein the monoclonal antibodies are produced from hybridoma cells
designated 4G11 having ATCC accession number HB11178 or antibody-producing
reclones thereof
reacting the monoclonal antibodies with a sample of rice tissue, spores or
extract thereof; and
measuring the specific binding reaction between the monoclonal antibodies
and Pyricularia grisea antigen in said sample to determine the presence of
Pyricularia grisea.
4. An immunoassay for the detection and identification of Pyricularia
grisea disease in rice crops, comprising the steps of:
reacting a sample of rice plant, spores from rice plant or extract thereof
from a rice crop with monoclonal antibodies specific for and highly
reactive with antigens of rice-pathogenic Pyricularia grisea race IB-49
highly reactive with Pyricularia grisea race IB-49 infected rice tissue,
substantially unreactive with healthy rice plant tissue, and weakly
reactive or unreactive with rice-or grass-pathogenic fungal genera other
than Pyricularia, wherein the monoclonal antibodies are produced from
hybridoma cells designated 4G11 having ATCC accession number HB11178 or
antibody-producing reclones thereof; and
measuring the specific binding reaction between said monoclonal antibodies
and Pyricularia grisea antigens in said sample to determine the presence
of Pyricularia grisea in the rice crop.
5. A hybridoma cell line producing antibodies specific for rice blast and
designated 4G11 having ATCC accession number HB11178 or antibody-producing
reclones thereof.
6. A monoclonal antibody specific for rice blast and produced by the
hybridoma cell line 4G11 having ATCC accession number HB11178 or
antibody-producing reclones thereof.
7. Apparatus for immunological diagnosis of Pyricularia grisea infection in
rice plants comprising:
a support surface;
monoclonal antibodies specific for and highly reactive with antigens of
rice-pathogenic Pyricularia grisea race IB-49, highly reactive with
Pyricularia grisea race IB-49 infected rice tissue, substantially
unreactive with healthy rice tissue and weakly reactive or unreactive with
rice-or grass-pathogenic fungal genera other than Pyricularia, wherein the
monoclonal antibodies are produced by hybridoma cells 4G11 having ATCC
accession number HB11178 or antibody-producing reclones thereof; and
means for measuring the specific binding reaction between the antibodies
and Pyricularia grisea antigens in a sample being diagnosed.
8. The apparatus as recited in claim 7 wherein said support surface is a
test tray including a plurality of test wells.
9. The apparatus recited in claim 7 further including a reagent supply for
facilitating the formation of an extract of rice plant tissue, spores or
mycelia to be tested.
10. A component of a monoclonal antibody-medicated enzyme-linked
immunosorbent assay kit for early detection and identification of
Pyricularia grisea comprising:
a solid substrate coated with a layer of monoclonal antibodies specific for
and highly reactive with antigens of Pyricularia grisea race IB-49 which
infect rice plants, highly reactive for Pyricularia grisea race IB-49
infected rice plant tissue, substantially unreactive with healthy rice
plant tissue and weakly reactive or unreactive with rice-or
grass-pathogenic fungal genera other than Pyricularia wherein said
monoclonal antibodies are produced by hybridoma cell line 4G11 having ATCC
accession number HB11178 or antibody-producing reclones thereof.
11. In an enzyme-linked immunosorbent assay test kit for detecting and
identifying Pyricularia grisea disease in rice crops, the improvement
comprising:
monoclonal antibodies specific for and highly reactive with antigens of
Pyricularia grisea race IB-49 which infect rice plants, highly reactive
for Pyricularia grisea race IB-49 infected rice plant tissue,
substantially unreactive with healthy rice plant tissue, and weakly
reactive or unreactive with other fungal genera found on rice plants
wherein said monoclonal antibodies are produced by hybridoma cells 4G11
having ATCC accession number HB11178or antibody-producing reclones
thereof.
12. In an immunofluorescence assay test kit for detecting Pyricularia
grisea disease in rice plants, the improvement comprising:
Monoclonal antibodies specific for and highly reactive with antigens of
Pyricularia grisea race IB-49 which infect rice plants, highly reactive
for Pyricularia grisea race IB-49 infected rice plant tissue,
substantially unreactive with healthy rice plant tissue, and weakly or
unreactive with rice-or grass-pathogenic fungal genera other than
Pyricularia wherein said monoclonal antibodies are produced by hybridoma
cells 4G11 having ATCC accession number HB11178 or antibody-producing
reclones thereof.
Description
BACKGROUND OF THE INVENTION
The present invention is directed to the serological identification and
detection of rice blast and, more particularly, concerns the development
of hybridomas which produce monoclonal antibodies specific for antigens of
the rice blast fungus, the monoclonal antibodies, and immunological
detection using the monoclonal antibodies.
Worldwide, fungi cause the greatest amount of crop damage as plant
pathogens and require the highest expenditures for their control. It is,
therefore, becoming critical that a rapid, reliable immunoassay for plant
pathogenic fungi is available to provide accurate diagnoses and
information about levels of particular fungi in the absence of disease
symptoms. Serological tests offer specificity, sensitivity, speed and
relative economy and have been applied to the detection of many plant and
animal pathogens in both clinical medicine and agriculture.
In contrast to immunoassays for plant viruses, progress in the development
of antibodies against fungi has been relatively slow because of the
difficulty in raising antisera that are specific. Until recently, fungi
could only occasionally be differentiated from closely related species on
the basis of their serological properties. Spores and mycelia of fungi
have many antigens in common, and conventional techniques used for
producing antiserum frequently developed complex mixtures of antibodies
(polyclonal antiserum) for the common antigens and rarely were able to
sort out the one or two specific antigens and develop antibodies for them.
However, the benefits of using monoclonal antibodies (MAbs) in the
diagnosis of diseases caused by fungi in agriculture, as well as in
clinical medicine, have been acknowledged recently. Antigens which have
been used successfully to produce specific MAbs against fungi include
soluble extracts of mycelia, mycelial homogenates, surface washings of
fungal solid cultures, extracellular components and cell wall materials of
fungi.
For example, U.S. Pat. No. 4,803,155 is directed to the development of
monoclonal antibodies which specifically bind to Sclerotinia homoeocarpa,
the causitive agent of dollar spot disease in plants; U.S. Pat. No.
4,845,197 discloses monoclonal antibodies adapted for the detection of
Phythiaceae infection of plants, U.S. Pat. No. 4,879,217 is directed to
monoclonal antibodies adapted for the detection of Rhizoctonia brown and
yellow patch; and U.S. Pat. No. 5,047,207 is directed to a kit for
diagnosing plant diseases and which employs monoclonal or polyclonal
antibodies specific for the suspected antigen.
Rice blast, caused by Pyricularia grisea Sacc., is a serious disease of
rice in many countries throughout the world. Rice blast rapidly changes
from minor to major importance with the potential for causing drastic
yield losses. This cyclic change usually occurs on a newly released rice
variety as a result of infrequently observed blast races pathogenic to the
new variety becoming widely established in commercial fields. As an
example, the Newbonnett rice variety, released in 1982, was observed to be
damaged by blast in 1985. A blast epidemic occurred in 1986 with at least
60,000 acres being severely damaged. In 1987, the disease developed early
in all rice production areas. Production costs for 1987 were increased by
subsequent fungicide applications. Regardless of the fungicide treatments,
many individual fields were severely damaged.
Blast moves from diseased plants to healthy plants in nearby fields by
airborne spores. Identical spores which do not infect rice are produced in
great numbers on many common grasses. A visual examination is not
sufficient to discriminate between the pathogenic and nonpathogenic
spores. Presently, flights of pathogenic spores can only be confirmed at a
minimum of five to six days later by the development of lesions on the
rice plant. By the time visible lesions have developed on the rice plants,
it may be too late to eradicate rice blast by the application of fungicide
and save the crop.
Commercial traps are available to catch spores for examination and
counting. These spore traps are used in Japan to predict blast epidemics
but have several limitations. Spores pathogenic to rice cannot be quickly
differentiated from the nonpathogenic spores from the grasses. The traps
are expensive and the predictive data based on the number of spores
trapped are applicable to the immediate area only. Japanese data on number
of spores caught and significance of spore flights would not necessarily
be applicable to other rice growing areas. Years of research are needed to
develop a forecasting system for a particular area.
Blast race identification is a difficult and time consuming process
consisting of isolating the blast fungus from diseased tissue or trapped
spores, culturing the fungus for spores, innoculating several differential
susceptible and resistant rice lines, and eventually determining the race
from differential lesion reactions among the rice lines. The entire
process may have to be repeated for closely related races. Current methods
for detecting the fungus to diagnose disease potential are laborious and
time-consuming.
Hence, there is a need for an improved method and apparatus for early
detection and identification of rice blast.
SUMMARY OF THE INVENTION
In accordance with the present invention, a method and apparatus for the
serological detection and identification of rice blast is provided which
includes the development and implementation of monoclonal antibodies
(MAbs) useful in identifying the rice blast pathogen. The present
invention includes the production of hybridoma cell lines which produce
MAbs specific for an extracellular component from an isolate of
Pyricularia grisea race IB-49.
The hybridoma technique for the production of specific antibodies allows
for differentiating between closely related antigens (blast spore races).
Single spleen cells from mice injected with antigens develop only one type
of antibody (monoclonal antibody). Thus, if the antibody is specific for
the one antigenic determinate that differentiates two races of rice blast
fungus, then the antibody can be used to identify a race within a short
time using a very simple serological test, Single spleen cells are not
long lived, but when fused with mouse myeloma cancer cells (forming a
hybridoma) they are immortal and will produce the one type of antibody
indefinitely.
In accordance with one example of the present invention, four monoclonal
antibodies (MAbs) were developed by fusing P3/NS1/1-Ag4-1 myelonia cells
with splenocytes from mice immunized with crushed conidial suspensions of
Race IB-49 of Pyricularia grisea. MAbs secreted from cell lines 4G11 (ATCC
deposited No. HB11178), 8H1 and 3E4 reacted mainly with conidial antigens,
showing MAb 4G11 bound on the surface of conidial cells whereas MAbs 8H1
and 3E4 bound on germ tubes in an immunofluorescence assay (IFA). MAb 11C6
reacted preferentially with mycelial antigen. Using ELISA, MAbs 8H1 and
3E4 showed partial cross-reactions with four unrelated fungal isolates and
MAb 11C6 with three from 11 genera tested. The species-specific MAb 4G11,
isotype IgG1, failed to recognize the antigens from the 11 fungal genera.
Further tests against 12 isolates of P. grisea or Pyricularia spp. from
rice or grasses indicated that MAb 4G11 reacted strongly with one,
moderately with two, weakly with five and negatively with four of the
isolates in IFA. MAb 4G11 could detect homologous conidial antigen at
15-70 ng per ml and 10-20 conidia per well by ELISA and appeared to have
potential diagnostic value.
Several monoclonal antibodies (MAbs) prepared against crushed conidia of
Pyricularia grisea Sacc. were characterized by using various immunological
assays as well as chemical and enzymatic analyses. MAb 4G11 recognized two
major proteins, one with an approximate molecular mass of 63 kilodaltones
(kDa) in crushed conidial suspensions, and the other about 20 kDa in
saline mycelial washings. Both proteins were present in sonicated mycelial
suspensions. The MAb 4G11 also bound to several minor proteins with
molecular weights ranging from 23-31 kDa in saline conidial washings.
Immunoelectron microscopy demonstrated binding of MAb 4G11 to the
cytoplasm of conidial cells and cytoplasm and walls of hyphal cells. It is
presumed that the 63 kDa protein was synthesized in the fungal cytoplasm
and then excreted as a smaller polypeptide. The epitopes recognized by
MAbs 8H1 and 3E4 were distributed mainly in conidial cytoplasm and on the
surface of growing points of germ tubes, whereas the the epitope to MAb
11C6 was present in both cell walls and cytoplasm of hyphae and conidia.
Chemical and enzymatic analyses confirmed that the epitope which reacted
with MAb 4G11 is either a protein or glycoprotein, and the epitopes which
reacted with MAbs 8H1, 3E3 and 11C6 are carbohydrates.
In accordance with one aspect of the present invention, monoclonal
antibodies specific for rice blast are utilized in onfarm test kits to
provide rapid detection and identification of rice blast. The most simple
test requires placing extracts of rice plant tissue or spores in contact
with the monoclonal antibodies and making a decision based on a color
reaction. The monoclonal antibodies of the present invention are also
adapted for use in spore traps for the purpose of differentiating between
spores pathogenic to rice and those pathogenic to grasses. Serological
tests that can rapidly differentiate between the races of blast are
invaluable to a blast race monitoring program and can be used to warn
growers of rapid changes or build-up of previously minor races on newer
established rice varieties.
In accordance with the present invention, three monoclonal antibodies
(MAbs) against Pyricularia grisea race IB-49 were produced and
characterized. Splenocytes from BALB/c mice immunized with an extract of a
liquid culture fluid were fused with NS-1 myeloma cells. The MAbs 3C3 and
4E10, isotype IgG3, and 10G9, isotype IgA, were characterized by indirect
ELISA with a variety of related and unrelated fungal isolates by Western
immunoblotting with antigen preparations of the fungus, and by
immunoelectron microscopy with the fungal culture and infected rice
tissue. All three MAbs reacted similarly with the antigens in various
immunoassay techniques used. The MAbs did not cross react with any
unrelated fungal isolates representing 11 genera, but reacted positively
with all 20 races or isolates of P. grisea. The MAbs bound to a high M
protein component (113kDa) in extracts of liquid culture fluids but not to
conidial antigens. The epitopes recognized by the MAbs were located in the
cell walls and lomasomes of hyphae with high density, and the cytoplasm of
conidia with low density. It is suggested that the antigenic component is
synthesized in the cytoplasm of fungal cells, accumulated in the lomasomes
and secreted through cell walls as an extracellular molecule. The MAbs
could detect homologous antigen at about 60 ng/ml fungal protein and
20-fold dilutions of the extracts of infected rice tissue by ELISA.
The principal object of the present invention,is the provision of a method
for serological detection and identification of rice blast. Another object
of the present invention is the provision of hybridoma cell lines which
produce monoclonal antibodies specific for antigens of the rice blast
fungus. A still further object of the present invention is the provision
of an immunoassay including monoclonal antibodies specific for the
detection of Pyricularia grisea. Yet another object of the present
invention is the provision of a spore trap including monoclonal antibodies
specific for antigens of rice blast. A more specific object of the present
invention is a method of detecting and identifying particular races of
Pyricularia grisea using monoclonal antibodies produced by hybridona cells
developed by fusing myeloma cells with splenocytes from mice immunized
with crushed conidial suspensions.
Other objects and further scope of the applicability of the present
invention will become apparent from the detailed description to follow
taken in conjunction with the accompanying tables and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a bar graph illustrating reactions of four MAbs from hybridoma
culture medium against antigens of Pyricularia grisea race IB-49 and rice
leaf tissue in ELISA;
FIG. 2 is a bar graph representing optical densities of three MAbs from
hybridoma culture when reacted with antigens of Pyricularia grisea race
IB-49 in ELISA;
FIG. 3 is a bar graph illustrating reactivity of MAbs 3C3 and 4E10 (ascites
fluid diluted at 1:5000) with isolates representing six races of
Pyricularia grisea in ELISA;
FIG. 4 is a bar graph representing reactivity of MAb 3C3 (ascites fluid
diluted 1:5000) with fungal isolates of Pyricularia grisea from rice and
grasses in ELISA;
FIG. 5 is a Western immunoblot analysis of MAbs against Pyricularia grisea
race IB-49. Fungal components separated by SDS-PAGE and transferred to a
nitrocellulose membrane were incubated with MAb (ascites fluid) and probed
with alkaline phosphatase-conjugated goat anti-mouse antibodies. Extract
of fluid from Pyricularia grisea liquid culture (lane 1-5): stained for
protein with Coomassie blue (1); immunostained with MAb 3C3 (2), 4E10 (3)
and 10G9 (4); immunostained with normal ascites fluid of mouse (5).
Crushed conidia (lane 6-7): stained for protein (6), immunostained with
3C3 (7). Saline conidial washing (lane 8-9): stained for protein (8), and
immunostained with MAb 3C3 (9);
FIG. 6a-6e are representations of immunogold labelling of fungal culture of
hyphae and conidia from fungal culture of Pyricularia grisea race IB-49
with MAb 3C3. The sections were incubated sequentially with MAb and
gold-conjugated goat anti-mouse antibodies prior to background staining.
Gold particles in hyphae were primarily located in the cell well (FIGS. 6a
and 6b) and in lomasomes, an extracellular space formed by invaginations
of the plasmalemma. In conidia, the gold particles occurred only in the
cytoplasm (FIG. 6c). When sections were incubated with MAb preabsorbed
with homologous antigen and normal ascites of mouse, no specific labeling
with gold particles occurred in hyphae (FIG. 6d) or conidia (FIG. 6e).
Scale bar=0.3 .mu.m;
FIG. 7a-7b are representations of immunogold labelling with MAb 3C3 of rice
tissue infected with Pyricularia grisea race IB-49. Specific labeling with
gold particles occurred only in the fungal cell wall even when the hyphae
was tightly pressed to the wall of host cells in either an early (FIG. 7a)
or late (FIG. 7b) stage of infection. A few scattered nonspecific
background gold particles are also shown. FC=fungal cytoplasm; HC=host
cytoplasm; FW=fungal cell wall; HW=host cell all. Scale bar=0.3 .mu.m;
FIG. 8 is a side sectional view of a spore trap in accordance with one
embodiment of the present invention;
FIG. 9 is a top sectional view of a spore trap in accordance with another
embodiment of the present invention;
FIG. 10 is a top sectional view of yet another embodiment of the present
invention;
FIG. 11 is a side sectional view of a test tray taken along line 11--11 in
FIG. 12;
FIG. 12 is a top plan view of a test tray in accordance with a kit
embodiment of the present invention;
FIG. 13 is a side view of a reagent bottle; and
FIG. 14 is a schematic side view of the test tray of FIGS. 11 and 12 being
examined under an ultraviolet light source.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with one embodiment of the present invention, conidial
antigens of P. grisea race IB-49 were used to produce MAbs specific for
the fungal antigens with the ultimate goal of developing a diagnostic
method to distinguish this fungus from other fungi found in rice fields.
Polyclonal antisera produced in rabbits were compared with MAbs.
MATERIALS AND METHODS
Isolation and culture of fungi
The isolates of races IB-49, IB-33, IC-17, IG-1, and IH-1 of Pyricularia
grisea were isolated at the Rice Research and Extension Center in
Stuttgart, Ak., USA and identified by lesion reactions on a series of
differential cultivars of rice. Slants of rice-polish agar (2 g agar and 2
g rice-polish in 250 ml of distilled water) were used for culture and
storage of the fungal isolates. For inoculum in the conidial production
process, yeast extract-glucose medium (3 g yeast extract and 15 g dextrose
in 1 L of distilled water) or rice-polish agar was used to grow mycelium
in shake and static cultures, respectively.
Sorghum grain was used for producing conidial spores of the isolates of
races IB-49, IB-33, IG-1, and IH-1. The grain was soaked, rinsed,
autoclaved, inoculated and incubated aseptically in flasks at 25-28 C
until the mycelia covered the grain. The fungus-covered grain was placed
on trays and incubated in a growth chamber with high relative humidity at
25-27 C under continuous cool-white fluorescent light. After 3-5 days, the
grain with the sporulating fungus was dried at 40 C for 24 h and
refrigerated in sealed plastic bags. The conidia were harvested from the
sporulated grain by vacuum.
Conidia of the isolate of race IC-17 were produced on corn leaves from 2-
to 3-weeks old plants (4-6 leaf stage). Leaves were cut in 10-15 cm pieces
and arranged on the surface of wet filter paper that had been placed in
the bottom of flasks or petri dishes. After being autoclaved, the leaves
were seeded with suspensions of either solid or liquid cultures of the
fungus and incubated at 25.degree. C. for 7-9 days until conidia covered
the pieces. Leaf pieces with conidia were dried and stored in a desiccator
at -20.degree. C. Conidia were collected by washing the pieces with
phosphate-buffered saline, pH 7.2, (PBS) and centrifuging at 1000 g.
Four isolates of P. grisea from grasses were obtained from Mississippi
State University, USA: PG #73 from crabgrass, PG #74 from ryegrass, PG
#89-1 from St. Augustinegrass, and PG #89-2 from millet. Isolate PG #15022
was furnished by the American Type Culture Collection. Five isolates of P.
grisea were isolated from rice fields in Arkansas. Rice-polish agar,
oatmeal agar, or V8 agar (3 g calcium carbonate, 200 ml V-8 juice and 12 g
agar in 1 L of distilled water) were used for culturing the isolates. Corn
leaves were used for producing conidia of the isolates.
Thirty-seven fungi representing 11 genera were isolated from rice-growing
areas in Arkansas. The genera were: Alternaria (six isolates), Curvularia
(seven), Helminthosporium (three), Fusarium (three) Aspergillus (three),
Penicillium (three), Monolinia (three), Rhizoctonia (four), Paecilomyces
(one), Pithomyces (one) and Cladosporium (three). All isolates were
cultured and maintained on potato-dextrose agar or oatmeal agar.
Preparation of germinating conidia for antigens
Conidia of isolates S-20 (race IB-49) and 75-A (race IC-17) were suspended
in PBS, 3-4.times.10.sup.5 /ml, and incubated in wells of 24-well tissue
culture plates (300 ul/well) at room temperature with shaking in the dark
for 16-18 h. The mixture of conidia and germinating conidia was
centrifuged at 1000 g for 10 min. Pellets were suspended in the same
volume of PBS, re-pelleted and used as antigens for immunization.
Preparation of crushed conidia for antigens
Conidia were suspended at a concentration of 10.sup.7 spores/ml in PBS and
washed three times by centrifugation at 1500 g for 10 min, first with PBST
(PBS containing 0.05% Tween 20), followed by two washes with PBS. After
the last wash, pellets were resuspended in PBS at a concentration of
2.times.10.sup.7 spores/ml and sheared by a French Press at cell pressures
of 2100-2800 kg/cm.sup.2.
Protein concentration of the antigens was determined by the BCA
(Bicinchonic acid, Pierce, Rockford, Ill., USA) protein assay with bovine
serum albumin (BSA) as the standard.
Preparation of polyclonal antisera
Polyclonal antisera for both races IB-49 and IC-17 were prepared in
rabbits. One rabbit for each race was immunized four times at weekly
intervals by subcutaneous injection of 2 ml of the germinating conidial
suspension without adjuvant. One week after the last injection, a blood
sample was taken from the marginal ear vein. Two or three months later, an
injection was given consisting of 2 ml of the antigen emulsified with
Freund's complete adjuvant. Bleedings were performed at intervals of about
two weeks.
Production of nonoclonal antibodies
Two Balb/c mice (5-7 weeks old) were given six intraperitoneal injections
of 3.5-4.5.times.10.sup.5 spores of the mixture of germinating conidia and
conidia in 500 ul PBS at 0, 2, 4, 6, 7, and 8 weeks and boosted with two
injections before fusion. Six mice were each injected with crushed
conidial suspensions (0.1 mg protein) mixed with 100 ul poly-A:poly-U (1
mg/ml in PBS; Sigma, St. Louis, Mo., USA). This was followed by 7
injections of 0.1-0.2 mg protein at two-week intervals and three booster
injections before fusion.
Mice were sacrificed by cervical dislocation, and the spleens were
suspended in 10 ml cell-PBS (PBS containing 0.2% sucrose) in a sterile
petri dish and disrupted on a sterile wire screen. Five ml of the
splenocyte suspension (1.times.10.sup.7 cells/ml) were mixed with the same
volume of actively growing myeloma cell line NS-1 (1.times.10.sup.7
cells/ml) and fused by the polyethylene glycol (PEG) method, (Groth, S. F.
De St., and Scheidegger, D. 1980. Production of monoclonal antibodies;
strategy and tactics. J. Immunol. Meth. 35:1-21). Cells were resuspended
in 24 ml of 10% FBS-HAT medium and plated out in 50 ul aliquots into five
96-well plates containing a macrophage feeder layer (3.6.times.10.sup.3
cells in 150 ul/well). After an incubation of 7-10 days, the wells
containing clones were identified and the culture fluids were assayed for
antibody production by indirect ELISA. Positive clones were chosen, and
culture fluids were further screened by ELISA and indirect
immunofluorescence assay (IFA). Selected cell lines were recloned 1-2
times by limiting dilution, grown in bulk, preserved by freezing slowly in
the culture medium/7.5% DMSO (Sigma) and maintained in liquid nitrogen.
Ascites production
For the production of ascites tumors, 5.times.10.sup.5 to 1.times.10.sup.6
hybridoma cells in a volume of 0.5 ml PBS were injected intraperitoneally
into BALB/c mice primed 10-14 days previously with 0.5 ml. of Pristane
(Sigma). After 12-16 days, the ascites fluid was withdrawn from the
peritoneal cavities with an 18-gauge needle and centrifuged at 400 g for
10 min. The ascites fluid was passed through a layer of cotton wool to
remove debris, fibrin clots and residual lipid and stored at -20.degree.
C.
Indirect ELISA
Wells of microtiter plates (Pro-bind, Beckton Dickinson, Lincoln Park,
N.J., USA) were coated (50-100 ul/well) overnight at room temperature with
antigens prepared in PBS, washed three times with PBST and once with
distilled water. Plates were used immediately or air-dried in a laminar
hood and stored in a plastic bag at 4.degree. C. for up to 4 weeks. Wells
were incubated at room temperature for 2 h with the supernatant fluid of
the hybridonia culture and then with peroxidase-conjugated goat anti-mouse
polyvalant (IgG+IgM+IgA) antibodies (Sigma) diluted in PBST. After
washing, color reaction was developed with the substrate (0.04%
0-phenylenediamine, 0.12% H.sub.2 O.sub.2 in citrate buffer, pH 5.0) and
read at 492 nm on an MR 600 Microplate reader (Fisher Scientific,
Pittsburgh, Pa., USA).
Indirect immunofluorescence assay
A drop of the conidial suspension (2-3.times.10.sup.4 spores/ml PBS) was
placed on a 22-mm diameter glass coverslip and incubated 5-6 h at
26.degree. C. in the dark for conidial germination. Coverslips were
washed, dried and stored at 4.degree. C. for up to 3 mo. Coverslips with
germinated conidia were blocked with PBS-BSA (PBS containing 1% bovine
serum albumin) for 30 min at room temperature and incubated sequentially
with detecting antibodies and FITC-conjugated goat anti-mouse polyvalent
(IgG+IgM+IgA) antibodies (Sigma). Coverslips were inverted onto glass
slides with a drop of mounting medium consisting of 80% glycerol, 0.1%
n-propylgallate and 0.1M Tris buffer, pH 8.6, and examined with an
epifluorescent microscope equipped with a general FITC filter set: BP
450-490, FT 500 and LP 515 (Olympus, Deer Park, N.Y., USA).
Controls were germinated conidia on coverslides treated by the same
procedure but omitting the step of antibody incubation or incubated with
hybridoma culture medium or normal ascites fluid instead of antibodies.
Determination of antibody isotype
A modified ELISA test was used to determine immunoglobulin isotypes. The
MAbs in supernatants of hybridoma cultures were captured in wells coated
overnight with fungal antigen specific for the antibodies. Captured
antibodies were incubated with rabbit anti-mouse antibodies specific for
one of the subclasses (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, and Kappa- and
Lambda-light chains) (Mouse Typer, Bio-Rad, Richmond, Calif., USA) for one
h and then probed with goat anti-rabbit peroxidase conjugates (Sigma).
Preparation of extracts from fungal culture and rice tissue
Fungal extracts used in ELISA were prepared by suspension of solid cultures
containing conidia and mycelia in PBS and mixing well with vortexing. The
slurry was centrifuged at 6000 g for 10 min, and the supernatant was used
for the assay.
Rice infection was carried out by inoculating rice plants (4-6 leaf stage)
with a conidial suspension (4.times.10.sup.5 spores/ml) and incubating in
greenhouse. Extracts of both infected and healthy rice tissue were made by
grinding rice leaves with mortar and pestle in PBS (1 g tissue/6 ml PBS),
filtering through Miracloth and centrifuging at 8000 g to remove insoluble
tissue debris.
RESULTS
Tests with polyclonal antisera
When rabbit polyclonal antisera raised against conidia of races IB-49 and
IC-17 were tested by ELISA against homologous and heterologous antigens,
both showed strong reactions with fungal extracts of both races and
cross-reactions with all unrelated fungi tested. Both antisera also
reacted with extracts of healthy as well as infected rice tissues.
IFA showed that the polyclonal antibodies bound mainly on the surface of
germ tubes and hyphae of the fungus. No binding on the conidial surface
was observed. A weak fluorescence was observed on the spore surfaces of
Aspergillus and Penicillium.
Monoclonal antibody agents conidia and germinating conidia
Twelve MAbs were developed using germinating conidia of race IB-49 as the
immunogen. Two of the MAbs, 2B5A1 and 2D1F1 (IgM class), were chosen for
further tests.
Analysis of antigenic components in conidial suspensions of race IB-49 by
ELISA indicated that the antigenic determinant specific for the MAbs was
present in the supernatant in relatively high concentration. IFA showed
that the epitopes were localized on the surface of conidia but not on germ
tubes and hyphae.
IFA with 2B5A1 and 2D1F1 also resulted in identification of cross-reactive
epitopes of conidia of three isolates of P. grisea, five isolates of
pyricularia sp., and one isolate each of Aspergillus sp., Penicillium sp.,
and Helminthosporium sp. No reactivity with five isolates of Pyricularia
sp, and seven genera of unrelated fungi was observed. In indirect ELISA
reactions occurred with extracts of healthy rice tissue and most of the
fungal isolates tested.
Production of monoclonal antibodies against crushed conidia
Three independent fusions were performed at different times. A summary of
the fusion results is given in TABLE I. In these tests, mice were
immunized with crushed spores of P. grisea race IB-49 produced in sorghum
grain culture. Because of the problem of cross-reaction of MAbs against
germinating conidia with rice tissue components, all the hybridomas in
these fusions were screened against both crushed conidial antigens (6 ug
total protein/ml) and extracts of healthy rice tissue (diluted 1:20).
Similar to the results obtained from the fusions with germinating conidia
as immunogen, most of the hybridoma supernatants positive for the
immunogen showed cross-reaction with rice plant components. Results
indicated that of 201 hybridomas, 117 reacted with both crushed conidia
and healthy rice leaf extract and 70 with rice leaf extract only. However,
14 of 201 cell lines (7%) secreted MAbs that were negative for rice leaf
extract, and from these, four stable cell lines were obtained (FIG. 1).
The four hybridomas, 4G11, 8H1, 3E4 and 11C6, were frozen in liquid
nitrogen, grown in bulk for further tests, and recloned. All the daughter
cell lines reacted the same as the parent cell lines.
In further assays with homologous antigens, 4G11, 8H1 and 3E4 reacted
strongly with the antigenic components of crushed conidia and conidial
suspensions and weakly with mycelial extract; 11C6 preferably recognized
mycelial antigen (FIG. 1).
Surface expression of the antigenic determinant recognized by the MAbs was
examined by IFA. The results revealed distinct differences in labelling on
different structures of P. grisea race IB-49. MAb 4G11 bound strongly to
the entire surface of conidial cells and only weakly or not at all to the
germ tubes. In contrast, bright fluorescence appeared on the surface of
the tip and basal areas of the germ tubes reacted with 8H1 and 3E4. No
fluorescence occurred on the middle area of the germ tubes or the conidial
surface. Only moderate fluorescence on hyphal pieces was found with MAb
11C6. No fluorescence was observed on the surfaces of the fungus treated
with hybridoma culture medium or normal mouse ascites.
The sensitivity of indirect ELISA was determined with undiluted culture
supernatant of hybridoma cell line 4G11 against homologous antigens of
race IB-49 (TABLE II). Results indicated that MAb 4G11 could detect the
antigenic component in supernatants of crushed conidia at protein
concentrations as low as 14-70 ng/ml and detected as few as 10-20
conidia/well.
Determination of isotype and titer of monoclonal antibodies
Isotyping of the MAbs showed that 4G11 was IgG1, 8H1 and 3E4 were IgG2a,
11C6 was IgM, and all clones were Kappa-light chain specific (TABLE III).
Indirect ELISA tests for antibody titer of cell supernatants ranged from
80-1280, while the titers of ascites fluids were 320,000, 1,280,000 and
2,560,000 for 4G11, 3E4 and 8H1, respectively (TABLE III). The titer
(40,000) of IgM MAb (11C6) was lower than the other three IgG class
antibodies. This may be due to lability of the antibody in culture
supernatant at 37.degree. C. incubation in addition to the nature of the
clone which might have produced antibodies for a minor antigenic
determinant of the antigen.
Cross-reactivity tests of MAbs
The four MAbs were tested further by ELISA and IFA against isolates
representing 11 genera of fungi occurring in rice fields (TABLE IV). In
ELISA, MAbs 8H1 and 3E4 reacted partially with four and weakly with one of
the fungal isolates and 11C6 reacted partially with three of the isolates.
However, MAb 4G11 did not give significant cross-reactions with isolates
of the 11 fungal genera isolated from rice plants in Arkansas. IFA
indicated that the four MAbs failed to bind to the surfaces of conidia of
10 of the fungi, and only 8H1 and 3E4 bound weakly to the surface of
hyphal pieces of the Rhizoctonia isolate.
For determination of cross-reaction of the MAbs with conidia of related
fungi, 17 isolates of P. grisea from both rice and grasses were tested by
IFA. When tested with MAb 4G11, strong fluorescence was found on the
conidial surface of isolate SUS #4, moderate fluorescence on that of SUS
3A and Kissi-1A (all three isolates belong to race IB-33), weak
fluorescence on eight other isolates and no fluorescence on the remaining
five isolates including race IC-17 (TABLE V).
Tests with MAb 4G11 against fungal extracts of 17 isolates of P. grisea by
ELISA are presented in TABLE V. In general the results of these assays
correlate with that of IFA, except race IC-17 which reacted strongly with
4G11 in ELISA but negatively in IFA. The isolates of race IB-33(Kissi-1A,
SUS-3A and SUS#4) showed a positive reaction with 4G11 in this test and
stained strongly or intermediately in IFA (TABLE V).
Detection of fungal antigen in infected tissue
PBS extracts of both infected and healthy rice leaf tissue were tested with
the MAbs by ELISA (TABLE VI). MAbs 8H1 and 3E4 did not recognize the
antigen in infected tissue, while 4G11 and 11C6 gave more than 6-fold
higher readings with extracts of infected tissue than with healthy tissue.
Discussion
Antisera to fungi are notoriously non-specific, Rabbit polyclonal antisera
raised against a mixture of conidia and germinating conidia of Pyricularia
grisea, races IB-49 and IC-17, demonstrated a lack of specificity.
In ELISA, polyclonal antisera raised against the two races of P. grisea
reacted strongly with fungi from very disparate genera and with PBS
extracts of healthy rice leaf tissue, It seems that most antigenic
components or epitopes that bind to the microtiter well surface may be the
most common and non-specific antigens. However, some differences in
binding ability of the polyclonal antisera on fungal structures were found
by IFA.
MAbs against germinating conidia of race IB-49 cross-reacted with most of
the fungal isolates representing 11 genera and reacted strongly with PBS
extracts of healthy rice tissue when tested by ELISA. The analysis of
antigens in spore suspensions by ELISA indicated the major antigenic
determinants specific for the MAbs are soluble components. It is suggested
that the epitope in the conidial suspension is an extracellular component
loosely associated with the cell surface which becomes soluble when
suspended in PBS. It is assumed that there are some readily soluble
homopolymers or heteropolymers of mannans in culture filtrates and in
supernatants of homogenized fungal cells. These polysaccharides frequently
have common determinants that contribute to cross-reactivity among fungi.
One of the key steps for the production of a MAb specific for fungal
antigen is the selection of the appropriate antigen. There is no consensus
in the literature regarding the type of immunogen likely to result in the
most specific antibodies. Teliospores of Tilletia species have been used
as antigens to produced MAbs and polyclonal antisera which did not exhibit
the desired level of specificity. However, cell wall antigens of
Aspergillus fumigatus have been used as immunogens to develop specific
MAbs. In accordance with the present invention, the assumption that
internal cell components might be more specific for fungal species led to
the use of crushed spores as antigen to produce MAbs.
In accordance with the present invention, conidia crushed by pressure
shearing is used as an immunogen, without Freund's adjuvant, and as a
source of antigen to screen hybridoma supernatants for developing MAbs
specific to fungal spores. Screening hybridomas against rice plant
components was necessary because, like the results obtained from fusions
with germinating conidia as immunogen, more hybridoma supernatants reacted
with extracts of rice leaf tissue than reacted with the immunogen.
However, four cell lines were chosen from 14 cell lines which were
negative for rice components. Supernatants from other cell lines were not
tested further because of their very low titer or their instability in
culture. The low titers could be caused by the outnumbering of specific
antigenic molecules binding to the wells by non-specific molecules in
ELISA.
In cross-reaction tests, MAb 4G11 did not react with heterologous fungal
isolates representing 11 genera in either ELISA or IFA. MAbs 8H1 and 3E4
reacted positively with four in ELISA and negatively with all of the
isolates using IFA. The reason for the different results from the two
assay methods may be that the main basis for the optical density reading
in ELISA was soluble components in the fungal extracts, whereas only the
components attached to the conidial surface contributed to the fluorescent
staining.
Tests by ELISA and IFA with homologous antigens showed that the MAbs
reacted differently with different fungal structures (FIG. 1). The results
indicated that the epitopes specific to the MAbs might be different
because of their different distributions on fungal structures.
A good correlation between the results of ELISA and of IFA was found when
MAb 4G11 was tested with 17 different isolates of P. grisea. The MAb
reacted positively with all of the predominant races, such as IB-49,
IC-17, IB-33, IH-1 and IG-1 in the United States. In IFA, a range of
staining degree (+++, ++, +or -) by the MAb 4G11 on conidia of the
different isolates of P. grisea may reflect the serological relationship
of the isolates with race IB-49.
From the above results, MAb 4G11, which is an IgG1 antibody, was designated
as a species-specific MAb. MAbs 8H1, 3E4 and 11C6, which cross-reacted
with some unrelated fungi in ELISA, are IgG2a and IgM. The results are
consistent with the belief that MAbs with highest specificity would be in
the IgG1 antibody subclass.
The capability of MAb 4G11 in detecting relatively small amounts of fungal
material (14-70 ng total protein/ml) and fungal pathogen in infected rice
tissue provides that MAb 4G11 can be used in diagnostic testing. When
testing the fungal antigens in diseased samples from rice fields in
Arkansas by ELISA, MAb 4G11 reacted positively with all 42 rice samples
infected with rice blast, and negatively with 11 other rice diseases
found. In accordance with the present invention, a detection kit would
include the MAb 4G11 either labelled with FITC for detection of conidia
using IFA, or conjugated with an enzyme for identification of the fungus
in extracts of contaminated rice seeds or of naturally infected rice
tissues using double antibody sandwich ELISA. Hybridoma cells of hybridoma
cell line 4G11 were deposited on or about Nov. 4, 1992 with the American
Type Culture Collection (ATCC), 12301 Parklawn Drive Rockville, Md. MD,
20852 USA and assigned ATCC accession number HB11178.
TABLE I
______________________________________
Summary of results of three fusions between myeloma
cells and splenocytes from mice immunized with crushed conidial
antigens of Pyricularia grisea race IB-49.
Fusion
I II III Total
______________________________________
Microplate wells seeded
960 1920 1440 4320
Wells with hybridomas screened
159 235 508 902
Hybridomas positive for the
15 47 69 131
immunogen
Hybridomas recognizing rice
21 45 121 187
tissue component
Hybridomas positive for the
3 4 7 14
immunogen and negative for
rice tissue
Stable cell lines grown in bulk,
1 2 1 4
frozen and recloned
Hybridomas mainly specific for
1 2 0 3
conidial antigen
Hybridoma mainly specific for
0 0 1 1
mycelial antigen
______________________________________
All cell lines were screened by indirect ELISA against the immunogen (6 ug
total protein/ml) and PBS extract of healthy rice leaf tissue (dilution
1:20).
TABLE II
______________________________________
Sensitivity of ELISA with culture supernatant of
hybridoma 4G11 against antigen of Pyricularia grisea race IB-49.
Crushed conidia.sup.a
A.sub.492nm.sup.b A.sub.492nm
Total protein/ml
Value Conidia/well.sup.c
Value
______________________________________
7000.0 >2.000 1250 >2.000
1400.0 0.562 625 1.008
700.0 0.435 313 0.578
140.0 0.317 156 0.501
70.0 0.252 78 0.267
14.0 0.104 39 0.264
7.0 0.107 20 0.205
1.4 0.046 10 0.149
PBS 0.044 PBS 0.052
______________________________________
.sup.a Conidia in PBS were sheared by a French Press. Values represent
ng/ml of total protein concentration.
.sup.b Value was the average of eight wells from two experiments.
.sup.c Conidia from sorphum grain culture were suspended in PBS.
Antigens coated in wells were incubated with undiluted culture fluid of
hybridoma cells and probed with peroxidaseconjugated goat anti-mouse
antibodies.
TABLE III
______________________________________
Isotyping and titer of monoclonal antibodies in
culture supernatant and ascites fluid
Elisa titer
Antibody Light- Culture Ascites
Cell line
isotype chain supernatant
fluid
______________________________________
4G11 IgG1 Kappa 640 320,000
8H1 IgG2a Kappa 1280 2,560,000
3E4 IgG2a Kappa 640 1,280,000
11C6 IgM Kappa 80 40,000
______________________________________
Coating protein concentration of homologous antigen in indirect ELISA was 6
ug/ml; ELISA titer is the reciprocal of highest dilution that gave an
A.sub.492nm value of three times larger than that of control (hybridoma
culture medium or normal mouse ascites).
TABLE IV
__________________________________________________________________________
Cross-reactivity of MAbs against antigens from 11 fungi
isolated from rice fields as determined by ELISA and IFA
Monoclonal antibodies
4G11 8H1 3E4 11C6
Organisma IFA.sup.a
ELISA.sup.b
IFA
ELISA
IFA
ELISA
IFA
ELISA
__________________________________________________________________________
P. grisea IB-49
+++
>2.000
- 1.964
- 1.842
- 0.387
Alternaria sp.
- 0.052
- 0.499
- 0.408
- 0.118
Aspergillus sp.
- 0.043
- 0.016
- 0.015
- 0.046
Cladosporium sp.
- 0.030
- 0.022
- 0.017
- 1.009
Curvularia sp.
- 0.033
- 0.117
- 0.101
- 0.018
Fusarium sp.
- 0.051
- 0.076
- 0.038
- 0.013
Helminthosporium sp.
- 0.090
- 0.615
- 0.409
- 0.043
Monilinia sp.
- 0.661
- 0.014
- 0.014
- 0.365
Paecilomyces sp.
- 0.029
- 0.016
- 0.018
- 0.031
Penicillium sp.
- 0.038
- 0.062
- 0.056
- 0.327
Pithomyces sp.
- 0.059
- 0.403
- 0.158
- 0.038
Rhizoctonia sp.
- 0.051
+ 0.514
+ 0.300
- 0.038
PBS (control)
- 0.048
- 0.054
- 0.035
- 0.067
__________________________________________________________________________
.sup.a Conidia on coverslips were incubated sequentially with MAbs and
FITCconjugated goat antimouse antibodies. Fluorescence on surface of
conidial cells: +++, strong; +, weak; -, none.
.sup.b Fungal extracts coated in wells were incubated with culture fluid
of hybridoma and probed with peroxidaseconjugated goat antimouse
antibodies. Total protein concentration ranged from 30-60 .mu.g/ml and 10
.mu.g/ml for race IB49. Values are average absorbance of 6 wells in two
assays at 492 nm.
All fungi were tested for reactivity with conidia in PBS (1.times.10.sup.3
-10.sup.6 spores/ml, depending on spore sizes), except Rhizoctonia in
which only hyphae were used for tests.
TABLE V
______________________________________
Reactivity of MAb 4G11 with fungal antigens of 17 isolates
of Pyricularia grisea in IFA and ELISA
ELISA.sup.c
Isolate Race.sup.a
Source IFA.sup.b
A.sub.492nm
Reac.
______________________________________
S-20 IB-49 Rice +++ 1.987 +
SUS 3A IB-33 Rice ++ 0.791 +
SUS #4 IB-33 Rice +++ 0.713 +
Kissi-1A IB-33 Rice ++ 0.645 +
75-A IC-17 Rice - 0.722 +
7412 IH-1 Rice + 0.636 +
7408 IG-1 Rice + 0.575 +
Katy G2N1
ND Rice + 0.323 +
Katy G2N2
ND Rice + 0.298 +
Katy G2N3
ND Rice - 0.067 -
Katy Law#4
ND Rice + 0.088 -
HWP/M201 ND Rice - 0.059 -
PG #89-1 ND St.Augustinegrass
+ 0.071 -
PG #89-2 ND Millet + 0.312 +
PG #73 ND Crabgrass + 0.267 +
PG #74 ND Ryegrass - 0.093 -
PG 15022 ND Grass - 0.112 -
______________________________________
.sup.a ND, not determined.
.sup.b Coverslips with germinating conidia were incubated with MAbs and
probed with FITCconjugated goat antimouse antibodies. Fluorescence: +++,
strong; ++, intermediate; +, weak; -, none.
.sup.c Total protein concentration of the fungal extracts ranged from 50
to 80 and 20 .mu.g/ml for race IB49. Antigen coated wells were treated
with MAb (undiluted culture fluid) and probed with peroxidaseconjugated
goat antimouse antibodies. Absorbance values at 492 nm are the average of
six wells in two tests. Reaction (Reac.) is determined as positive (+) if
the values are three times larger than that of control (PBS), otherwise a
negative (-).
TABLE VI
______________________________________
Detection of fungal antigen in infected rice tissue.sup.a
with MAbs by indirect ELISA
Absorbances.sup.b
MAbs Infected
Healthy
______________________________________
4G11 0.289 0.041
8H1 0.117 0.056
3E4 0.108 0.062
111C6 1.078 0.049
______________________________________
.sup.a Samples were extracted by grinding rice leaf tissues (1 g) in 5 ml
PBS and centrifuging.
.sup.b Mean values of 2 tests, each with 3 replicate wells. Samples coate
in wells were incubated with MAbs (ascites fluid diluted at 1:3000-4000)
and probed with peroxidaseconjugated goat antimouse antibodies.
FIG. 1. Reactions of four MAbs from hybridoma culture medium against
antigens of Pyricularia grisea race Ib-49 and rice leaf tissue in ELISA.
Total protein concentration for crushed conidial suspension, conidial
suspension and PBS extract of fungal mycelia was 10 ug/ml and for PBS
extract of rice leaf tissue 100 ug/ml. Antigen coated wells were treated
with MAbs (undiluted culture fluid) and probed with peroxidase-conjugated
goat anti-mouse antibodies. Absorbance values (492 nm) are the average of
6 wells from two tests.
In accordance with another example of the present invention, Mabs specific
for rice blast are produced by hybridoma lines created by immunizing mice
with extracts of liquid culture fluid.
Fungi and fungal culture
Fungal isolates representing P. grisea races IB-49, IB-45, IB-33, IC-17,
IG-1, IH-1 were obtained from tile Rice Research and Extension Center in
Stuttgart, Ak. Five field isolates of P. grisea were isolated from rice in
Arkansas. Five isolates of P. grisea from grasses were furnished by
Mississippi State University, USA. Rice-polish agar (2 g agar and 2 g
rice-polish in 250 ml of distilled water) and oatmeal agar were used for
maintaining and culturing the isolates, and autoclaved sorghum grains were
used as the medium for producing conidia of the fungus.
Thirty-seven fungal isolates representing 11 genera were isolated from rice
plants in rice-growing areas of Arkansas. All isolates were cultured and
maintained on potato-dextrose agar or oatmeal agar.
Preparation of immunogen
Liquid culture of the fungus was carried out in the following medium: 10 g
of rice-polish, 2 g of yeast extract, 1 g of (NH.sub.4).sub.2 SO.sub.4,
0.5 g MgSO.sub.4.7H.sub.2 O per 1 liter of distilled water. The broth was
inoculated with 10-15 ml of a mycelial suspension of liquid culture or a
petri dish of slab culture and cultured by shaking at
26.degree.-28.degree. C. The culture was incubated for 5-6 days until the
appearance of a gray pigment and then filtered twice through a layer of
Miracloth (Calbiochem Co., La Jolla, Calif. USA). The filtrate was
centrifuged at 18,000 g for 20 min. The resulting supernatant of fungal
liquid culture was used to screen hybridoma cells during antibody
production. Further concentration was achieved by acetone precipitation
and lyophilization. Twice the volume of cold acetone was added to the
solution, allowed to stand at -20.degree. C. for one h, and centrifuged at
4,000 g for 10 min. The pellets were kept under vacuum for 5 min to
eliminate residual acetone, dissolved in a small amount of 0.1M phosphate
buffer (PB), pH 7.2, and centrifuged to remove the insoluble materials.
The extract was lyophilized, and the resulting extract was resuspended in
phosphate-buffered saline (PBS) before use. Protein concentration of the
antigen preparations was determined by the BCA (bicinchoninic acid) method
(Pierce, Rockford, Ill., USA).
Preparation of antigens for specific test of Mabs
Five fungal antigenic sources were prepared from an isolate of P. grisea
race IB-49: (1), saline mycelial washings: mycelia growing on sorghum
grains were washed with 2-4 volumes of PBS, and the resultant supernatant
was further concentrated by acetone precipitation and lyophilization; (2),
saline conidial washings were made using the same method as that of
mycelial washings; (3), sonicated mycelia were obtained by sonicating a
fungal mycelial suspension for 5 min in an ice bath at one min intervals;
(4), crushed conidia were prepared by shearing the conidia suspended in
PBS at 10.sup.8 spores/ml with a 40K French-Press Cell Press (AMCO) at a
cell pressure of 2500 kg/cm.sup.2 ; (5), extracts of both infected and
healthy rice tissue were made by grinding rice leaves with mortar and
pestle in PBS (1 g tissue/6 ml PBS), filtering through a layer of
cheesecloth and centrifuging at 6000 g to remove insoluble tissue debris.
The resulting supernatants were stored at -20.degree. C. and diluted in
PBS before tests.
Immunization
Six- to 8-weeks-old female BALB/c mice were immunized intraperitoneally
with 150 ng of antigen (extract of liquid culture fluid) treated by
(AL).sub.3 OH precipitation plus two drops of heat-killed Bordetella
pertussis cells. On days 14, 28, 42, 56 and 70, 180 ug of the same antigen
in saline was injected intraperitoneally without adjuvant. Two weeks after
the final injection, the mice were bled from the tail, and antisera were
screened by enzyme linked immunosorborent assay (ELISA) against the fungal
culture fluid and rice plant tissue. Selected mice received three booster
injections at 3-4 day intervals before fusion.
Fusion and MAb production
Three days after the final booster injection, mice were sacrificed by
cervical dislocation, and spleens were removed. Cells of the myeloma cell
line NS-1 and splenocytes were fused by a modified procedure described by
Groth, S. F. De St., and Scheidegger, D. 1980. Production of monoclonal
antibodies: strategy and tactics. J. Immunol. Meth. 35:1-21. The mixture
of myeloma cells and splenocytes was suspended in 30% (w/v) polyethylene
glycol (PEG, MW 4000) in serum-free Iscoves MD medium (Sigma, St. Louis,
Mo., USA) for 2 min at 37.degree. C. The cell pellet was slowly overlaid
with 40 ml of the medium and allowed to stand for 5 min. PEG was removed
by centrifugation, and the pellet was washed once with the medium. The
cells were resuspended in 24 ml of 10% FBS-HAT (fetal bovine serum and
hypoxanthine, aminopterin and thymidine) Iscoves MD medium (Sigma), and
plated out into five 96-well plates.
After 10-14 days, the wells containing clones were assayed for antibodies
by ELISA against both liquid culture fluid of a race IB-49 isolate of P.
grisea and extract of infected rice leaf tissue. Selected cell lines were
recloned twice by limiting dilution, grown in bulk in non-selective
medium, preserved by freezing slowly in FBS/DMSO (dimethyl sulfoxide) and
maintained in liquid nitrogen. For production of MAb, pristane-primed
BALB/c mice were injected intraperitoneally with 10.sup.6 hybridomas per
mouse. Ascites fluid was drained 10-15 days later and screened for
antibody production by ELISA.
ELISA
Screening of hybrid clone culture supernatant for antibodies against
antigen was carried out by indirect ELISA. Wells were coated overnight at
room temperature with liquid culture fluid, extract of rice tissue and
other antigens. Wells were incubated for 2 h with the primary antibody, 1
h with peroxidase-conjugated goat anti-mouse IgG or polyvalent
(IgG+IgM+IgA) antibodies (Sigma, St. Louis, Mo., USA) diluted in PBST
(PBS+0.05% Tween 20), and 20-30 min with the substrate (0.04%
o-phenylenediamine, 0.012% H.sub.2 O.sub.2 in 0.1M citrate buffer, pH
5,0). All washes between incubations were done with PBST. The reaction was
stopped by adding 3M H.sub.2 SO.sub.4, and read at A.sub.492 nm on a MR
600 microplate reader (Dynatech, Chantilly, Va., USA) that had been
blanked against an empty well. Wells were considered positive if they had
an absorbance reading greater than 3 times the control wells incubated
with culture medium in place of the hybridoma culture fluid.
Determination of immunoglobulin isotype
A modified ELISA test was used for isotyping of MAbs. MAbs were captured in
wells coated overnight at 4.degree. C. with homologous antigen. The wells
were incubated with rabbit anti-mouse antibodies specific for one of the
subclasses IgG1, IgG2a, IgG2b, IgG3, IgM, IgA and Kappa- or Lambda-light
chain (Mouse Typer, Bio-Rad, Richmond, Calif.) for 1 h, and then probed
with peroxidase-conjugated goat anti-rabbit antibodies (Sigma, St. Louis,
Mo., USA).
SDS-PAGE and western blotting
SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting
techniques were conducted by a modified method described previously in
Hearn, V. M., Wilson, E. V., Latge, J. P., and Mackenzie, D. W. R. 1990.
Immunochemical studies of Aspergillus fumigatus mycelial antigens by
polyacrylamide gel electrophoresis and Western blotting techniques. J.
Gen. Microbiol. 136:1525-1535, and Mencke, N., Donoghue, P. O. Lumb, R.,
Smith, P., Tenter, A. M., Thummel, P., and Rommel, M. 1991. Antigenic
characterization of monoclonal antibodies against Sarcocystis muris by
Western blotting and immuno-electron microscopy. Parasistol. Res.
77:217-223. The samples were electrophoresed through a 5% stacking gel and
a 10% resolving gel (vertical gel system) at 200 V for 45-60 min under
reducing conditions in a minigel system (Bio-Rad, Richmond, Calif., USA).
The denaturing sample buffer contained 0.125 M Tris-HCL (pH 6.8), 2% (w/v)
SDS, 10% glycerol, 5% (v/v) 2-mercaptoethanol, and 0.004% bromphenol blue.
Prestained SDS-PAGE molecular mass standards (15-110 kDa; Bio-Rad) were
included in each gel. The gel was cut into two pieces. One of the pieces
was stained with Coomassie blue to visualize protein bands. The proteins
in the other piece of the gel were transferred to a nitrocellulose
membrane (pore size 0.2 um, Bio-Rad) using a TE 22 Mini Transphor system
(Hoefer Sci. Inst., San Francisco, Calif., USA) at 200 mA for 2 h.
After blocking with 4% defatted milk overnight at 4.degree. C., the
membrane was subsequently incubated on a shaker for 2 h at room
temperature in ascites fluid diluted in PBST, and 1 h with a solution of
alkaline phosphatase-conjugated goat anti-mouse IgG or polyvalent
(IgG+IgM+IgA) antibodies (Sigma, St. Louis, Mo., USA). Washes between
incubations were performed for 20 min with four changes of PBST. The color
reaction was developed by incubating with 10 ml substrate solution
containing 3.5 mg bromochloroindolyl phosphate (BCIP) and 1.75 mg nitro
blue tetrazolium (NBT) and stopped by rinsing with 20 mM EDTA-PBS followed
by distilled water.
Immunoelectron microscopy
Young rice plants were inoculated by spraying them with a conidial
suspension in PB (4.times.10.sup.5 spores/ml) and incubating in a plastic
bag at high relative humidity (RH) for 24 h and then in the greenhouse for
4-6 days. Samples for electron microscopy were 1-2 mm.sup.2 pieces of
symptomatic leaf tissue taken from the border of lesions. Samples of the
rice blast fungus growing on rice-polish agar were cut into 1 mm.sup.2
blocks.
Both the agar blocks and infected leaf pieces were fixed in a modified
Karnovsky's fixative consisting of 2% glutaraldehyde and 2%
paraformaldehyde in 0.05M cacodylate buffer at pH 7.0 for at least 4 h.
Conidial suspensions were centrifuged at 1000 g, and resulting pellets
were also fixed in the same fixative for 24 h. Post-fixation was omitted
or conducted in 1% osmium tetroxide for 2 h. The specimens were embedded
in Spurr's low viscosity medium (de Souza, V. B. V., Gergerich, R. C.,
Kim, K. S., and Langham, M. A. C. 1990. Properties and cytopathology of a
tymovirus isolated from eggplant. Phytopathology 80:1092-1098) and
polymerized in a 70.degree. C. oven overnight.
Immunogold labelling was carried out according to a modified procedure
described by de Souza, et al. Sections were incubated on nickel grids in
PBS containing 1% bovine serum albumin (PBS-BSA) for 30 min, The grids
were washed with PBST, and incubated with ascites fluid (MAbs) diluted in
BSA-FBS-PBST (PBST containing 0.1% BSA and 5% fetal bovine serum) for one
h. After washing 4.times.5 min with PBST, the grids were either incubated
In a small drop (50 ul) of 10 nm colloidal gold particle-conjugated goat
anti-mouse IgG or polyvalent (IgG+IgM+IgA) antibodies (Sigma, St. Louis,
Mo., USA) diluted 1:25 in BSA-FBS-PBST for 45 min. The grids were washed
with PBST and rinsed with distilled water. Finally, the grids were stained
with 2% aqueous uranyl acetate and lead citrate for 10 min and 5 min.
respectively, and examined in a JEOL-100 CX transmission electron
microscope.
The controls used to determine the specificity of the gold labelling
included: omission of the MAb incubation in the standard procedure, use of
normal ascites fluid in place of the MAbs in the procedure, and adsorption
of MAb with homologous antigen before incubation.
Results
MAb Production and characterization
Culture fluids from 178 hybridomas were screened 12 days after fusion
against both liquid culture fluid of the fungus and an extract of rice
leaf tissue by indirect ELISA. Nine (5%) of 178 hybridoma cell lines
secreted MAbs that were positive for the fungal antigen and negative for
the extract of healthy rice tissue. From these, three stable cell lines
were chosen for further investigation (TABLE VII). The cloned cell lines,
3C3, 4E10, and 10G9 were propagated in BALB/c mice for ascites fluid
production. The titers of ascites fluids ranged from 1:160,000-1:320,000
and those of cell culture supernatants were 1:320-1:640 in indirect ELISA
(TABLE VII). MAbs 3C3 and 4E10 belonged to the murine IgG3 isotype
subclass, whereas MAb 10G9 was IgA, and all had a Kappa light chain. In
sensitivity tests, the MAbs could detect the extracellular component of
fungal proteins in the extract of liquid culture fluid at about 60 ng
total protein/ml (TABLE VII).
Reactivity of MAbs in ELISA
Relative reactivities of MAbs with different preparations of P. grisea race
IB-49 isolate were detected in indirect ELISA (FIG. 2). The three MAbs
reacted strongly with sonicated mycelia and saline mycelial washes as well
as with the extract of liquid culture fluid. The MAbs reacted weakly with
crushed conidia and saline conidial washing.
In ELISA assays with extracts of both infected and healthy rice leaf
tissue, the MAbs gave 4-9 fold higher optical readings with the extract of
infected tissue than with that of healthy tissue (TABLE VIII) at a 1:5
dilution of tissue extracts in PBS.
Cross-reactivity of the MAbs was tested against unrelated fungi isolated
from plants in rice-growing areas in Arkansas representing 11 genera
(TABLE IX). None of the three MAbs, 3C3, 4E10 and 10G9, showed significant
cross-reactions with any of the 11 fungal isolates (TABLE IX).
For determination of reactivity of the MAbs with related fungi, the
following fungal isolates were tested using ELISA: Race IB-49, IB-45,
IC-17, IH-1, IG-1 and IB-33 (isolates SUS 3A, SUS #A, Kissi-1A) of P.
grisea from the Rice Research and Extension Center (Stuttgart, Ak.); four
isolates of P. grisea from Mississippi State University, PG 73 from
crabgrass, PG 74 from ryegrass, PG 89-1 from St. Augustinegrass, and PG
89-2 from millet; one isolate PG #15022 furnished by the American Type
Culture Collection; and five isolates of Pyricularia spp., named Katy
G2N1, N2, N3, Katy Law#4 and HWP/M201 isolated from ricegrowing areas in
Arkansas. The three MAbs, 3C3, 4E10 and 10G9, reacted positively with all
twenty fungal isolates cultured in rice-polish agar or oatmeal agar. Some
quantitative differences in optical readings were, however, found among
the isolates (FIGS. 3 and 4). Reactivity of each of the three MAbs with
the fungal isolates was similar. Only the test results of MAbs 3C3 and
4E10 with different races (FIG. 3) and MAb 3C3 with ten isolates from rice
or grasses (FIG. 4) are shown here.
SDS-PAGE and Western blot
In Western blots of SDS-PAGE gels of the extract of liquid culture fluid of
the P. grisea race IB-49 isolate, the same major protein band was detected
with all three MAbs, 3C3, 4E10 and 10G9 (FIG. 5, lane 2-4). The protein
was a large molecule with an M of 113 kDa and was not recognized by normal
mouse ascites fluid (lane 5). Two conidial preparations, French
Press-crushed conidia and saline conidial washes of the fungus, were also
analyzed by Western blotting. No visible protein band was detected in
either conidial antigen preparation by the three MAbs (lane 7 and 9).
However, a protein band (113 kDa) was observed in saline conidial washes
stained with Coomassie blue (lane 8). The same result occurred when the
test was repeated two more times. This result may imply that the protein
molecule found in conidia is identical in size, but may be different in
structure or conformation.
Immunoelectron microscopy with gold labelling
Similar patterns of the epitope distribution in fungal cells of the P.
grisea race IB-49 isolate were observed among MAbs 3C3, 4E10 and 10G9 by
immunoelectron microscopy with gold labelling. Typical results with MAb
3C3 are shown in FIGS. 6 and 7. Immunolabelling of the fungus from the
culture medium resulted in gold particles being associated with the cell
wall (FIG. 6A, B) or lomasome (FIG. 6B), an extracellular space formed by
the invagination of the plasmalemma. In contrast, in conidia, the gold
particles occurred only in the cytoplasm (FIG. 6C). When the fungal
sections were incubated with normal ascites fluid or the MAbs absorbed
with the respective homologous antigen, no specific labelling with gold
particles occurred (FIG. 6D, E).
Immunogold labelling of rice leaf tissue infected with P. grisea the race
IB-49 isolate resulted in the gold particles specific to each of the three
MAbs being located in the cell wall of fungal hyphae in the host tissues
(FIG. 7A, B). Immunogold labelling also revealed that MAb 3C3 was specific
for an epitope which was located in developing young conidia and
conidiophores which were growing out through a stoma. In cases where a
fungal hypha was surrounded by necrotic host cells and the cell walls of
the host and the pathogen were adhering tightly to each other, the gold
particles were still located only in the fungal wall indicating MAbs'
specificity.
DISCUSSION
Previous research has confirmed that the key step for the production of a
MAb specific for particular organisms is the selection of the appropriate
antigen preparation. There is no consensus in the literature regarding the
type of immunogens likely to result in the most specific antibodies. In
accordance with the present invention, it has been shown that the
cell-free extract of liquid culture fluid can be successfully used as the
immunogen and as a source of antigen to screen hybridoma supernatants for
producing MAbs specific for the fungal antigens of P. grisea. The fact
that all three MAbs selected from 178 clones produced by two fusions
reacted positively with all the races or isolates of P. grisea tested, but
negatively with 11 genera of unrelated fungi, indicates the production of
MAbs specific to P. grisea that can be used for reliable diagnostic
purposes.
The specificity of MAbs depends on the source of immunogen used, and the
degree of difficulty in raising MAbs expressing a particular specificity
of interest has been suggested to depend on both the levels of soluble
smaller proteins and the presence of non-specific carbohydrates or
glycoproteins that induce a non-T cell stimulated response in mice. The
fact that success was achieved with hybridomas developed against culture
extracts indicates that the extracellular components in liquid culture
fluid may be more specific and Immunogenic as an antigen source of the
fungus for production of MAbs than intracellular components. It is also
possible that treatment of the antigenic preparation by ammonium sulfate
precipitation might be beneficial in terms of immunogenicity because the
non-specific carbohydrates and other smaller components could be removed
from the preparation. An added benefit is that removal of carbohydrates
reduces the possibility that less desirable immunoglobulins such as IgM
are produced.
The source of antigen from a fungus could result in the production of MAbs
which are specific for different structures in the same fungus. In
accordance with the present invention, MAbs raised from an extracellular
component in fungal liquid cultures reacted strongly with hyphal
preparations as well as the extract of culture fluid and very weakly with
conidial antigens in ELISA. Interestingly, similar results of reactivity
were observed among the MAbs in various serological tests against the
antigens. Western immunoblotting clearly demonstrated that the three MAbs,
3C3, 4E10 and 10G9, bound to the same major protein component with a high
Mr of 113 kDa, suggesting that the extracellular protein or polypeptide
might be highly immunogenic in mice. However, the epitopes recognized by
the three MAbs may or may not be the same. The extracellular protein was
not detected in conidial antigen preparations by the MAbs in Western
blotting probably because the 113 kDa protein was different in structure
or conformation.
Although the site and nature of specific antigens are not generally known,
locations and expressions of epitopes in or on fungal tissues are reported
for some MAbs prepared against fungi. In the present examinations of P.
grisea cultures with immunoelectron microscopy and gold-labelling, the
epitope(s) recognized by the MAbs were located in the cell walls and
lomasomes with high density gold deposition and in the cytoplasm of the
hyphae with low density deposition. The epitope(s) were only located in
the cytoplasm of conidia with low density gold deposition. As the cell
grows, the component may accumulate in the lomasomes followed by secretion
through the cell wall into its environment (culture medium). The high
density of gold-labelling in fungal cell walls suggests that the component
is associated with cell walls by either staying there temporarily during
passage or becoming a constitutive component.
Examination of sections of infected leaf cells revealed various cytopathic
effects caused by the invading fungus. Several molecular components
produced by P. grisea showing phytotoxic effects or mediating infective
processes have been characterized.
The sensitivity in ELISA tests of the MAbs with extracts of liquid culture
fluids was approximately 60 ng/nil total protein in the extracts of liquid
culture fluids and 1:5 dilution of the extract of infected rice leaf
tissue. As such, the MAbs can be used in a diagnostic detection kit for P.
grisea in infected or contaminated rice tissues.
TABLE VII
______________________________________
Isotyping, titer and sensitivity in ELISA of
monoclonal antibodies against extract of culture fluid from
cultures of Pyricularia grisea
ELISA Titers.sup.a
ELISA.sup.b
Antibody Light- Culture Ascites
Sensitivity
Cell Line
isotype chain supernatant
fluid (ng/ml)
______________________________________
3C3 IgG3 Kappa 640 320,000
60-120
4E10 IgG3 Kappa 320 160,000
60-120
10G9 IgA Kappa 320 320,000
12-60
______________________________________
.sup.a Coating protein concentration of homologous antigen in indirect
ELISA was 12 ug/ml; ELISA titer is the reciprocal of the highest dilution
that gave A.sub.492 nm value of three times larger than that of controls
(hybridoma culture medium or normal ascites of mouse). Values shown are
the means of two tests, each with five replicate wells.
.sup.b MAbs (ascites) were diluted 1:15,000 in PBStween; sensitvity of
ELISA is the lowest protein concentration of antigen that gave A.sub.492
nm value three times larger than that of control (PBS). Values shown are
the means of two tests, each with five replicate wells.
TABLE VIII
______________________________________
Detection of Pyricularia grisea antigen in rice leaf tissue.sup.a
with monoclonal antibodies (MAbs) by ELISA
A.sub.492 nm.sup.b
MAbs Infected
Healthy
______________________________________
3C3 0.391 0.039
4E10 0.383 0.048
10G9 0.254 0.061
______________________________________
.sup.a Samples were extracted by grinding 1 g of rice leaf tissue in 6 ml
of PBS and centrifuging. Supernatants were diluted at 1:5 in PBS before
tests.
.sup.b Mean values of 2 tests, each with 3 replicate wells. Wells were
coated with samples and incubated with MAbs (ascites fluid diluated at
1:3000-4000) and probed with peroxidaseconjugated goat antimouse
antibodies.
TABLE IX
______________________________________
Cross-reactivity of MAbs against antigens from eleven
fungi isolated from rice fields as determined by ELISA
Monoclonal antibody.sup.b
Organism.sup.a 3C3 4E10 10G9
______________________________________
Alternaria sp. 0.020 0.019 0.018
Aspergillus sp.
0.050 0.037 0.023
Cladosporium sp.
0.091 0.088 0.055
Curvularia sp. 0.026 0.022 0.024
Fusarium sp. 0.025 0.020 0.023
Helminthosporium sp.
0.040 0.034 0.028
Monilinia sp. 0.021 0.019 0.032
Paecilomyces sp.
0.065 0.024 0.020
Penicillium sp.
0.021 0.021 0.018
Pithomyces sp. 0.023 0.026 0.035
Rhizoctonia sp.
0.069 0.072 0.056
Pyricularia grisea
1.907 1.853 1.387
IB-49
Fungal culture medium
0.028 0.023 0.015
______________________________________
.sup.a All fungi were tested for reactivity with fungal culture (mycelia
and conidia) diluted in PBS. Total protein concentration ranged from 30 t
60 ug/ml, and 10 ug/ml for P. grisea race IB49.
.sup.b Antigencoated wells were incubated with undiluted supernatants of
hybridoma cultures (MAbs) and probed with peroxidaseconjugated goat
antimouse antibodies. Values are average absorbance of 6 wells of two
assays at 492 nm.
FIG. 2. Optical densities of three MAbs from hybridoma culture when reacted
with antigens of P. grisea race IB-49 in ELISA. Protein concentration for
extract of liquid culture fluid (ELCF), sonicated mycelia (SMY), saline
mycelial washings (SMW), crushed conidia (CRC) and saline conidial
washings (SCW) was 6 ug/ml. Medium=hybridoma culture medium. The
antigen-coated wells were incubated sequentially with MAbs and
peroxidase-conjugated goat anti-mouse antibodies. Values are the averages
of 9 wells of three assays of 492 nm.
FIG. 3. Reactivity of MAbs 3C3 and 4E10 (ascites fluid diluted at 1:5000)
with isolates representing six races of P. grisea in ELISA. Total protein
concentration of the antigens (mycelial and conidial suspensions) prepared
from the fungal races was 20 ug/ml. Absorbance values are the average of 6
wells from two tests.
FIG. 4. Reactivity of MAb 3C3 (ascites fluid diluted 1:5000) with fungal
isolates of P. grisea from rice and grasses in ELISA. Fungal isolates from
rice: race IB-49 (49), Katy G1N1 (N1), G1N2 (N2), G1N3 (N3), Katy Law#4
(#4) and HWP/M201 (201); Isolates from grasses: PG 73 (73), PG 74 (74), PG
89-1 (9-1), PG 89-2 (9-2) and PG 15022 (22). Total protein concentration
of the mycelial and conidial suspension was 30 ug/ml. Absorbance values
are the average of 8 wells of two tests.
FIG. 5. Western immunoblot analysis of MAbs against Pyricularia grisea race
IB-49. Fungal components separated by SDS-PAGE and transferred to a
nitrocellulose membrane were incubated with MAb (ascites fluid) and probed
with alkaline phosphatase-conjugated goat anti-mouse antibodies. Extract
of fluid from P. grisea liquid culture (lane 1-5): stained for protein
with Coomassie blue (1); immunostained with MAb 3C3 (2), 4E10 (3) and 10G9
(4); immunostained with normal ascites fluid of mouse (5). Crushed conidia
(lane 6-7): stained for protein (6), immunostained with 3C3 (7). Saline
conidial washing (lane 8-9): stained for protein (8), and immunostained
with MAb 3C3 (9).
FIGS. 6A-6E, Immunogold labelling of hyphae and conidia from fungal culture
of Pyricularia grisea race IB-49 with MAb 3C3. The sections were incubated
sequentially with MAb and gold-conjugated goat anti-mouse antibodies prior
to background staining. Gold particles in hyphae were primarily located in
the cell wall (FIGS. 6A and 6B), and in lomasomes, an extracellular space
formed by invaginations of the plasmalemma (FIG. 6B). In conidia, the gold
particles occurred only in the cytoplasm (FIG. 6C) When sections were
incubated with MAb pre-absorbed with homologous antigen or normal ascites
of mouse, no specific labelling with gold particles occurred in hyphae
(FIG. 6D) or conidia (FIG. 6E). Scale bar=0.3 .mu.m.
FIGS. 7A-7B Immunogold labelling with MAb 3C3 of rice tissue infected with
Pyricularia grisea race IB-49. Specific labelling with gold particles
occurred only in the fungal cell wall even when the hyphae was tightly
appressed to the wall of host cells in either an early (FIG. 7A) or late
(FIG. 7B) stage of infection. A few scattered nonspecific background gold
particles are also shown. FC=fungal cytoplasm; HC=host cytoplasm;
FW=fungal cell wall; HW=host cell wall. Scale bar=0.3 .mu.m.
As shown in FIG. 8 of the drawings, and in accordance with one embodiment
of the present invention, a spore trap generally designated by the
reference numeral 10 is shown to include an inlet 12, a settlement chamber
14, including a plurality of baffles 16, and a housing 18. Housing 18
therein supports a rotating drum 20 having attached thereto a spore
collecting support or substrate 22, having a moist, tacky, reactive layer
24 including monoclonal antibodies specific for antigens of rice blast.
Layer 24 and support 22 are, for example, an agar impregnated fibrous
material backed by a sturdy plastic material. A vacuum source 26 is
attached to housing 18. The spore trap 10 is adapted for use in the rice
field to detect rice blast during critical plant growth stages. Activation
of vacuum source 26 draws airborne constituents including rice blast
spores into the inlet 12. Settlement chamber 14 collects debris other than
spores so as to reduce the contaminants and other objects which contact
and adhere to the reactive monoclonal antibody layer 24 of the support 22,
A filter 28 serves to stop the passage of spores and other debris so as to
protect the vacuum source 26. Once spores have been collected on the
support 22, it is removed from the spore trap 10 and analyzed under a
microscope to count the number of spores, and, examined for a color change
that would indicate the presence of rice blast spores. Next, the spores
are allowed to germinate for a given allotment of time, and then the layer
24 is examined again visually and under ultraviolet light so as to detect
a color change in the monoclonal antibody layer 24 due to the presence of
any pathogenic rice blast spores. Preferably, vacuum source 26 is a
battery powered vacuum source such as a small, electric, battery powered
reed pump or motor and fan arrangement. Drum 20 is rotated by a small
electric motor 30 mounted on the base of housing 18. Preferably, electric
motor 30 is battery operated.
As illustrated in FIG. 9 of the drawings and in accordance with another
embodiment of the present invention, a spore trap generally designated by
the reference numeral 30 is shown to include an inlet 32, a cylindrical
housing 34, a cylindrical fine screen 36, a spore collecting substrate 38
having a moist, tacky, reactive layer 40 including monoclonal antibodies
specific to rice blast antigen, and a rotating drum 42. Screen 36 and
housing 34 define therebetween a primary settlement chamber. The interior
of drum 42 defines a secondary settlement chamber. Drum 42 includes an
inlet 44, a filter 46, and an outlet 48. Outlet 48 is connected to a
vacuum source 50, via a conduit 52. Filter 46 serves to block the passage
of spores and other debris and thereby protect vacuum source 50.
Cylindrical screen 36 and substrate 38 are connected to drum 42 and, as
such, rotate with drum 42 under the power of, for example, a battery
operated motor. Vacuum source 50 is preferably a battery powered vacuum
source including, for example, a small electric motor and fan assembly.
Spore trap 30 is designed to be placed in the field and to draw in spores
and other debris through inlet 32 while screen 36 and substrate 38 are
rotated. The primary settlement chamber collects debris and other airborne
material drawn in through inlet 32. Spores pass through screen 36, impinge
upon, and adhere to the monoclonal antibody layer 40 of substrate 38.
Following collection of spores, substrate 38 is removed from the spore
trap 30 so that the spores may be counted and the layer 40 examined for a
color change. After a germination period, layer 40 is examined again
using, for example, an ultraviolet light source to detect any color change
which would indicate the presence of pathogenic rice blast spores.
As represented in FIG. 10 of the drawings and in accordance with yet
another embodiment of the present invention, a spore trap generally
designated by the reference numeral 60 is shown to include an inlet 62, a
cylindrical housing 64, and a rotating drum 66. Housing 64 and drum 66
define therebetween a settlement chamber 68. Drum 66 includes an inlet 70,
a support bracket 72, a spore collecting substrate 74 attached to bracket
72 and having a moist, tacky, reactive, monoclonal antibody, front layer
76, a filter 78, and an outlet 80. Outlet 80 provides fluid communication
to a vacuum source 82 connected thereto by a conduit 84. Vacuum source 82
is preferably a battery powered vacuum means including, for example, a
small electric motor and fan assembly. Spore trap 60 is designed to be
placed in the field and upon activation of vacuum source 82 and rotation
of drum 66 by, for example, a small, battery powered, electric motor,
spores and other debris are drawn in through inlet 62 into the interior of
housing 64. The heavy debris tends to collect in settlement chamber 68
while lighter debris and spores pass through opening 72 and impinge upon
and adhere to the monoclonal layer 76 of substrate 74. Filter 78 serves to
protect vacuum source 82 by blocking the passage of small debris and
spores. After spores have been collected on the reactive layer 76,
substrate 74 is removed from the spore trap 60 for evaluation. The spores
are counted and reactive layer 76 is examined under, for example, a source
of ultraviolet light to detect and identify pathogenic rice blast spores.
After a short germination period, the reactive layer is examined again for
color changes indicative of the presence of rice blast.
In accordance with still yet another embodiment of the present invention,
and as shown in FIGS. 11-14 of the drawings, a field test kit for
detecting and identifying rice blast in rice plant tissue, conidia,
mycelia or spores includes a test tray generally designated by the
reference numeral 100, a pair of small pipettes 102 and 104, a bottle of
reagent 106, and an ultraviolet light source 108.
Test tray 100 is formed of a rigid plastic material, such as polyvinyl
chloride, or of a ceramic material and Includes a large extract well 110
and four smaller test wells 112, 114, 116 and 118 which interrupt an
otherwise substantially planar upper test tray surface 120. Each of the
test wells 112 through 118 is coated on its inner surface with a reactive
layer including monoclonal antibodies specific for a particular pathogenic
rice blast race, for example, layer 122 in well 118. Extract well 110
serves as a container and grinding surface for the rice plant tissue,
conidia, mycelia or spores to be tested for pathogenic rice blast. A cap
124 on reagent bottle 106 is shaped so as to be used as a pestle in
conjunction with the large well 110 for grinding of the rice plant tissue,
conidia, mycella or spores following the addition of a small amount of
reagent from the bottle 106. Following grinding of the rice plant tissue,
conidia, mycelia or spores in the reagent to form an extract to be tested,
a few drops of extract is placed in each of the test wells 112-118 using
one of the small pipettes 102 and 104. Pipettes 102 and 104 are preferably
sterile glass tubes having a small inner diameter which provides for
capillary action to fill the pipette with extract while tending to prevent
the drawing up of debris. Pipettes 102 and 104 are secured to the upper
surface 120 of test tray 100 by two strips of transparent tape 126 and
128,
Ultraviolet light source 108 is preferably a hand-held battery powered
ultraviolet light source including a short fluorescent bulb 130 which
emits ultraviolet light. Following a short incubation period, the presence
of pathogenic rice blast in the extract tested is determined by analyzing
each of the test wells for positive reactions using ultraviolet light
source 108 (FIG. 14).
In accordance with another embodiment of a test kit in accordance with the
present invention, each of the test wells 112 through 118 would include an
assay layer incorporating a monoclonal antibody specific for an antigen of
pyricularia grisea race IB-49, such as monoclonal antibodies produced by
hybridoma cell lines 4G11 (ATCC deposited No. HB11178), 3C3, 11C6, and
4E10. Also, it is contemplated that one of the test wells may be coated
with a control layer which does not include antibodies. It is contemplated
that the test trays 100 would be shipped in air-tight sterile packages
which would not be opened until ready for use in the field.
The immunoassay processes and techniques of the present invention provide
for the early detection and identification of the rice blast fungus using
hybridoma lines which secrete monoclonal antibodies specific for the blast
fungus pyricularia grisea. The preferred hybridoma line is 4G11 which
secretes monoclonal antibodies specific for P. grisea, but not with
contaminating fungi or healthy plant tissue.
The use of the test kits and spore traps of the present invention utilizing
monoclonal antibodies specific for rice blast antigen is much more
accurate and faster than current methods of relying on symptom development
or identification in the field, or transporting samples to a trained
diagnostician for identification purposes.
The present invention encompasses the use of detection kits, spore traps,
and serological systems to detect spore movement into and within a
specific area, such as a rice field or larger production area. The
monoclonal antibodies of the present invention may also be used to detect
and quantify the fungus in rice seed. Further, the monoclonal antibodies
of the present invention can be used to measure blast disease levels in
order to predict disease severity and establish economic thresholds for
disease control efforts.
In accordance with the present invention, monoclonal antibodies such as
MAbs 3C3, 4E10 isotype IgG3, 10G9 isotype IgA, 4G11, 8H1, and 3E4 are
adapted for use in immunoassay apparatus, processes, and techniques, and
in spore traps, processes, and techniques to provide early detection and
identification of the rice blast fungus in rice plant tissue, conidia,
mycelia or spores. For example, race specific monoclonal antibodies can be
utilized in kits to provide rapid identification of a particular race of
rice blast in a particular rice crop. The most simple test consists of
placing extracts of diseased plant tissue in contact with the monoclonal
antibodies and making a decision based on a color reaction, Such a product
provides an on-farm test that confirms plant symptoms as being rice blast.
Also, the race specific monoclonal antibodies can be used in spore traps
for the purpose of differentiating between spores pathogenic to rice and
those pathogenic to grasses, The serological tests can rapidly
differentiate between the races of blast and, as such, are invaluable to a
blast race monitoring program and can be used to warn growers of rapid
changes or buildup of previously minor races on new or established rice
varieties.
Hybridoma cell lines 4G11, 11C6, 3C3, 8H1, 3E4, 4E10, and 10G9 are
currently stored at the Hybridoma Lab of,the Biotechnology Center at the
University of Arkansas, Fayetteville, Ak., U.S.A.
In accordance with yet another aspect of the present invention, DNA RFLPs
were used to analyze genetic variation in the rice blast pathogen
(Magnaporthe grisea) population on a microgeographic scale. One hundred
and thirteen isolates were collected from two rice fields (cv. Newbonnet)
in Arkansas. In addition, several reference isolates representing the
predominant races in Arkansas were also examined. Total DNA of each
isolate was cut with EcoRI and probed with a dispersed repeated "MGR" DNA
probe (Hamer et al. PNAS, 1989; Levy et al., The Plant Cell, 1991).
Isolates were scored for similarity based on the presence or absence of
approximately 50 DNA fragments ranging in size from 2-20kb. Based on DNA
similarities, seven distinct fingerprint groups were identified. Isolates
within a group had >80% shared fragments and <50% shared fragments between
groups. Of the seven groups identified (A through G), only four (A, B, C,
and D) were identified in the two field populations. Group A was the
predominant group found representing 72% and 53% of the Isolates collected
in the two fields. Groups B and D were similar to (approx. 80% shared
fragments) two of the reference strains (group B=race IG-1, lineage IG-1B;
and group D=race IC-17, lineage IC-17, Levy et al.). Groups C and E were
similar to lineages IB-49A and IB-49B, respectively (Levy, et al.). Field
isolates, representing the four groups (A, B, C, and D) identified in the
two fields as well as several reference isolates, were compared for
virulence in greenhouse pathogenicity tests.
Thus, it will be appreciated that as a result of the present invention, a
highly effective method and apparatus for serological detection and
identification of rice blast is provided by which the principal objective,
among others, is completely fulfilled, It is contemplated, and will be
apparent to those skilled in the art from the preceding description and
accompanying drawings, that modifications and/or changes may be made in
the illustrated embodiments without departure from the present invention,
Accordingly, it is expressly intended that the foregoing description and
accompanying drawings are illustrative of preferred embodiments only, not
limiting, and that the true spirit and scope of the present invention be
determined by reference to the appended claims.
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